A new withanolide from the roots of Withania somnifera - NOPR

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Several oximes and oxime ethers have been developed as antimicrobial agents. A series of chlorooximes (hydroximoyl chlorides) have been synthesized and ...

Indian Journal of Chemistry Vol. 47B, May 2008, pp. 740-747

Biologically active hydroxymoyl chlorides as antifungal agents† Tabasum Ismaila, Syed Shafia, Parvinder Pal Singha, Naveed Ahmed Qazia, Sanghapal D Sawanta, Intzar Alib, Inshad Ali Khanb, H M S Kumar*a, Ghulam Nabi Qazid & M Sarwar Alamc a Departments of Synthetic Chemistry & bBiotechnology, Department of Chemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi, 110 062

c d

Indian Institute of Integrative Medicine (formerly RRL, Jammu), Canal Road, Jammu Tawi 180 001, India E-mail: [email protected] Received 12 July 2007; accepted (revised) 19 February 2008

Several oximes and oxime ethers have been developed as antimicrobial agents. A series of chlorooximes (hydroximoyl chlorides) have been synthesized and tested for antifungal activity under in-vitro conditions against Candida albicans, Candida parapsilosis, Candida glabrata, Candida krusei, Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger. The derived antifungal activity has been compared with the corresponding oximes. The results show that most of the chlorooximes exhibit potent antifungal activity with anti-isomers showing better activity. It is observed that most of the chlorooximes show interesting antifungal activity (MICs < 32 µg/mL) compared to oximes. Compound 3q (2,3-dimethoxy phenyl hydroxymoyl chloride) is the most active compound. This compound is active against all the Candida species (MIC 0.5 µg/mL) as well as filamentous fungi with MIC range of 2-4 µg/mL. This series of compounds are fungicidal in nature as evident from the MFC results. Keywords: Hydroxymoyl chloride (chlorooxime), oximes, hydroxylamine hydrochloride, N-chlorosuccinimide, antifungal activity

It is well known that fungi cause many diseases of plants, animals, and humans and often acquire drug resistance during treatment. Since fungal infections are caused by eukaryotic organisms, for this reason they generally present more difficult therapeutic problems than do bacterial infections. Fungal infections have emerged as a major cause of morbidity and often of mortality in immunocompromised and debilitated patients over the past two decades1,2. Many of the currently available drugs are toxic, produce recurrence because they are fungi static and not fungicidal or lead to the development of resistance due in part to the prolonged periods of administration of the available antifungal drugs. The usage of most antimicrobial agents is limited, not only by the rapidly developing drug resistance, but also by the unsatisfactory status of present treatments of bacterial and fungal infections and drug side-effects3-6. Although the use of a new generation of triazoles, the available polyenes in lipid formulations, the use of echinocandins or the combination therapy have been introduced as alternatives in the last ten years, fungal ⎯⎯⎯⎯⎯⎯ † IIIM Communication No. SCL-07/18

infections remain difficult to eradicate7. There is, therefore, a clear need for the discovery of new chemical entities with antifungal properties, which could lead to the development of new drugs for the management of fungal infections. Oximes and their derivatives have attracted considerable attention since the past few decades due to their chemotherapeutic value. Many oximes are found to be antihyperglycemic8, anti-neoplastic9, anti-inflammatory10, anti-leishmanial11, and VEGFR-2 kinase inhibitors12. Oximes also possess transcriptional activity13. Besides this, several oximes and oxime-ethers have been developed as anti-microbial agents14. The current study attempted to assess particularly the antifungal effects of various chlorooximes on different strains of fungi such as Candida albicans, C. parapsilosis, C. glabrata, C. krusei, Aspergillus fumigatus, A. flavus and A. niger to explore their therapeutic potential. Chemically, chlorooximes are important intermediates for the synthesis of nitrile oxides which in turn are used in a number of chemical reactions such as dipolar cyloaddition reactions and lead to the synthesis of a variety of heterocycles like isoxazoles, isoxazolines, etc. Even though oximes were used as

ISMAIL et al.: HYDROXYMOYL CHLORIDES AS ANTIFUNGAL AGENTS

pharmacophoric groups for the generation of highly effective anti-microbials, no efforts has been hitherto made to explore the biological potential of their halogenated analogs, i.e., chorooximes. In the present paper, is reported the synthesis of a focused library of chlorooximes to evaluate their antifungal activity including minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) against standardized ATCC isolates along with an analysis of the structure-activity relationship (SAR) in comparison to the corresponding parent oximes. Results and Discussion Chemistry Herein is reported a library of oximes and their corresponding chlorooximes acting as antifungal agents. The oximes and chlorooximes were synthesized as per the literature procedure (Table I) (ref. 15). To the neutralized solution of hydroxylamine hydrochloride, aldehyde 1 was added and the reaction mixture was stirred for 1 hr at RT. Excess of water was added to the reaction-mixture and organic compound was extracted with ethylacetate (2×50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to afford pure oxime (syn and anti) in 99% yield. Oximes 2, when treated with N-chlorosuccinimide in DMF led to the synthesis of corresponding chlorooximes 3 in good yields (>85%). All these synthesized compounds (Table I) were screened for anti-fungal activity against seven fungal strains (Candida albicans, Candida parapsilosis, Candida glabrata, Candida krusei, Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger) using microdilution technique. Antifungal activity and structure activity relationship studies The MIC and MFC of the oximes and chlorooximes is described in Table II. Among the main observations, it can be stated that most of the chlorooximes showed interesting antifungal activity (MICs < 32 µg/mL) whereas oximes showed no such attractive MICs. Among these derivatives, compounds 3a, 3b, 3i, 3m, 3q, 3r, 3s, 3t and 3v showed significant activity against C. albicans, C. parapsilosis, C. glabrata, C. krusei, A. fumigatus, A. flavus and A. niger with MICs in single digits. Compound 3q was the most active compound. This compound was very potent against all the Candida species (MIC

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0.5 µg/mL). It was also active against filamentous fungi with MIC range of 2-4 µg/mL. This series of compounds were fungicidal in nature as evident from the MFC results. SAR studies on these compounds revealed that oximes are less potent than chlorooximes. Among chlorooximes, compound 3q (2,3-dimethoxy phenyl hydroximoyl chloride) exhibited highest activity (MIC 0.5 µg/mL and MFC 0.5 µg/mL), showed to be the most potent fungicides amongst all the substrates studied and was active against all the strains of Candida and Aspergillus species. Even though a variety of chlorooximes derived from phenyl, substituted phenyl and heteroaromatic oximes exhibited potent antifungal activity, both electron donating and electron withdrawing groups on aromatic nucleus have shown no appreciable effect on the MIC and MFC values. Similarly, bulky aromatic rings like napthyl and anthracyl oximes did not have profound effect on the antifungal activity. Chlorooximes derived from aliphatic oximes have shown lower activity in comparison with their aromatic counterparts. Since these compounds exist in two isomeric forms i.e, syn- and anti-isomers, the need to examine the antifungal activity of each isomeric form was felt. Thus, compound 3q being the most potent antifungal derivative, was subjected to column chromatography (silica gel 230-400 mesh as stationary phase, hexane/ethylacetate as mobile phase) and both geometrical isomers were isolated in pure form. Both the isomers were identified on the basis of their coupling constant values, syn-isomer having coupling constant values of 8.01 Hz and 8.06 Hz whereas anti-isomer having coupling constant values of 8.93 Hz and 8.94 Hz. The distinction between syn and anti could also be made on the basis of polarity (syn being more polar than anti) and their respective melting point differences (syn-isomer having melting point of 108.8oC and anti-isomer having melting point of 92oC). Each isomer thus isolated was screened for anti-fungal activity against the above mentioned seven strains (Table III). It was found that anti-isomer was more potent than synisomer. It is now well established that the zinc and calcium dependent family of proteins called the MMPs (matrix metalloproteinases) which are secreted by fungus such as Candida albicans, hydrolyses the collagen proteins on skin and consequently causes fungal infections under physiological conditions. These are selectively regulated by endogenous inhibitors.

INDIAN J. CHEM., SEC B, MAY 2008

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Table I — Synthesis of various oximes and their corresponding chlorooximes H

NH2OH.HCl

RCHO

2a 3a

130 50

2b 3b

133 102

2c 3c

100-02 95-98

2d 3d

146-48 142-45

2e

127-28.5

3e

151-52

2g

R

2 m.p (oC)

R

2l 3l

3g

2h

85-87

3h

72-73

2i 3i

96-97 76-77

2j 3j

119-20

2m

74-75

3m

71.5-72.5

2n 3n

107-08 153-54

MeO

2o 3o

89-91 89

MeO

2p 3p

63 49

H3C

N

NC

CH3

N H

Me2N

F

Cl

2q 3q

96.5 111.8-13

2r

111-12

3r

126-27

2s 3s

98 97

2t 3t

147-4 semisolid

2u

157 semisolid

3u

99-100

OMe OMe O O

HO HO

F

114.5-15 104-06

R

liquid 50

NO2

2k 3k

syn / anti

m.p (oC)

NO2

197-200 180

144 semisolid

3

Compd

S

OH N

R

1

2f 3f

Cl

NCS,DMF

N

NaOH/H2O

Compd

OH

2v 3v F

a) Products characterised by IR, 1H NMR and mass spectral analysis

146.5 114-15

ISMAIL et al.: HYDROXYMOYL CHLORIDES AS ANTIFUNGAL AGENTS

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Table II — The minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) of oximes, chlorooximes and the control drug. MIC and MFC are expressed in µg/mL Entry

Test organisms C. albicans

Amphotericin-B 2a 3a 2b 3b 2c 3c 2d 3d 2e 3e 2f 3f 2g 3g 2h 3h 2i 3i 2j 3j 2k 3k 2l 3l 2m 3m 2n 3n 2o 3o 2p 3p 2q 3q 2r 3r 2s 3s 2t

Yeast C.parapsilosis C. glabrata

C. krusei

A. fumigatus

Filamentous Fungi A. flavus

A. niger

MIC 0.5

MFC 0.5

MIC 0.5

MFC 1.0

MIC 0.5

MFC 0.5

MIC 1.0

MFC 1.0

MIC 0.5

MFC 1.0

MIC 1.0

MFC 1.0

MIC 0.5

MFC 1.0

>64 4.0 >64 8.0 >64 >64 >64 64 16 32 >64 >64 >64 >64 >64 32 >64 8.0 >64 64 >64 32 >64 16 >64 8.0 >64 >64 >64 >64 >64 >64 >64 1.0 >64 16 >64 8.0 >64

>64 4.0 >64 8.0 >64 >64 >64 >64 32 64 >64 >64 >64 >64 >64 32 >64 8.0 >64 >64 >64 32 >64 32 >64 8.0 >64 >64 >64 >64 >64 >64 >64 1.0 >64 16 >64 8.0 >64

>64 2.0 >64 4.0 >64 >64 >64 64 16 32 >64 >64 >64 >64 >64 16 >64 8.0 >64 64 >64 16 >64 16 >64 4.0 >64 >64 >64 >64 >64 >64 >64 1.0 >64 8.0 >64 8.0 >64

>64 4.0 >64 8.0 >64 >64 >64 >64 16 32 >64 >64 >64 >64 >64 32 >64 8.0 >64 >64 >64 32 >64 16 >64 8.0 >64 >64 >64 >64 >64 >64 >64 1.0 >64 8.0 >64 8.0 >64

>64 2.0 >64 4.0 >64 >64 >64 64 16 32 >64 >64 >64 >64 >64 16 >64 8.0 >64 64 >64 16 >64 16 >64 4.0 >64 >64 >64 >64 >64 >64 >64 1.0 >64 8.0 >64 8.0 >64

>64 4.0 >64 8.0 >64 >64 >64 64 16 32 >64 >64 >64 >64 >64 32 >64 8.0 >64 >64 >64 16 >64 16 >64 4.0 >64 >64 >64 >64 >64 >64 >64 1.0 >64 8.0 >64 8.0 >64

>64 2.0 >64 4.0 >64 >64 >64 64 8.0 32 >64 >64 >64 >64 >64 16 >64 4.0 >64 64 >64 16 >64 8.0 >64 2.0 >64 >64 >64 >64 >64 >64 >64 0.5 >64 4.0 >64 4.0 >64

>64 2.0 >64 4.0 >64 >64 >64 64 16 32 >64 >64 >64 >64 >64 16 >64 4.0 >64 64 >64 16 >64 16 >64 2.0 >64 >64 >64 >64 >64 >64 >64 0.5 >64 4.0 >64 4.0 >64

>64 8.0 >64 32 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 64 >64 16 >64 64 >64 32 >64 32 >64 16 >64 >64 >64 >64 >64 >64 >64 4.0 >64 32 >64 16 >64

>64 8.0 >64 64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 64 >64 16 >64 >64 >64 32 >64 32 >64 16 >64 >64 >64 >64 >64 >64 >64 4.0 >64 32 >64 16 >64

>64 4.0 >64 32 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 64 >64 8.0 >64 64 >64 32 >64 16 >64 16 >64 >64 >64 >64 >64 >64 >64 2.0 >64 16 >64 16 >64

>64 4.0 >64 32 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 64 >64 16 >64 >64 >64 32 >64 16 >64 16 >64 >64 >64 >64 >64 >64 >64 2.0 >64 32 >64 16 >64

>64 4.0 >64 16 >64 >64 >64 64 64 >64 >64 >64 >64 >64 >64 32 >64 16 >64 64 >64 16 >64 16 >64 8.0 >64 >64 >64 >64 >64 >64 >64 2.0 >64 16 >64 16 64

>64 4.0 >64 16 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 >64 32 >64 16 >64 >64 >64 32 >64 16 >64 8.0 >64 >64 >64 >64 >64 >64 >64 2.0 >64 16 >64 16 >64 ⎯ Contd

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Table II — The minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) of oximes, chlorooximes and the control drug. MIC and MFC are expressed in µg/mL ⎯ Contd Entry

Test organisms Yeast C. albicans

Amphotericin-B 2t 3t 2u 3u 2v 3v

C.parapsilosis

Filamentous Fungi C. glabrata

C. krusei

A. fumigatus

A. flavus

A. niger

MIC 0.5

MFC 0.5

MIC 0.5

MFC 1.0

MIC 0.5

MFC 0.5

MIC 1.0

MFC 1.0

MIC 0.5

MFC 1.0

MIC 1.0

MFC 1.0

MIC 0.5

MFC 1.0

>64 4.0 >64 >64 >64 4.0

>64 8.0 >64 >64 >64 8.0

>64 4.0 >64 >64 >64 4.0

>64 4.0 >64 >64 >64 4.0

>64 4.0 >64 >64 >64 4.0

>64 4.0 >64 >64 >64 4.0

>64 2.0 >64 >64 >64 2.0

>64 2.0 >64 >64 >64 2.0

>64 16 >64 >64 >64 16

>64 16 >64 >64 >64 16

>64 8.0 >64 >64 >64 16

>64 16 >64 >64 >64 16

64 8.0 >64 >64 >64 8.0

>64 16 >64 >64 >64 8.0

Table III — The comparative MIC and MFC values for each isomer of compound 3q. MIC and MFC are expressed in µg/mL. Test organisms

Entry Yeast C. albicans 3q syn 3q anti

Filamentous Fungi

C. parapsilosis

C. glabrata

C. krusei

A. fumigatus

A. flavus

A. niger

MIC 4.0

MFC 4.0

MIC 4.0

MFC 4.0

MIC 4.0

MFC 4.0

MIC 2.0

MFC 2.0

MIC 8.0

MFC 16

MIC 8.0

MFC 8.0

MIC 8.0

MFC 8.0

1.0

1.0

1.0

1.0

1.0

1.0

0.5

1.0

4.0

8.0

4.0

8.0

4.0

8.0

Imbalances between the active enzymes and their natural inhibitors lead to the fungal disease. The potential for using specific enzyme inhibitors to redress this balance has led to intensive research focused on the design, synthesis16, and molecular deciphering of low molecular mass inhibitors of this family of proteins. Moreover, certain derivatives, such as oximes and hydrazides, also possess selective chelating or binding properties with the zinc activesite of MMPs. Such small molecule MMP inhibitors act either as competitive substrates or distort the geometry of one of zinc centers in MMPs by binding with such zinc cations in the form of a five or sixmember ring with one or two double bonds, respectively, in a bidentate structure form. After distorting the geometry of such zinc cations, these MMP inhibitors appear to move away from this "deactivated" active-site and go to the next active-site to deactivate it17. Thus, a similar mechanism of antifungal action is envisaged by chloroximes and oximes as they are strong ligands for zinc binding. A detailed mechanistic evaluation is currently in

progress to investigate the actual role of this novel class of anti-fungal compounds. Materials and Methods Antifungal activity of all compounds was performed using microdilution method (NCCLS M27 A, NCCLS M38 P) against four yeast strains (Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019, Candida glabrata ATCC 90030, Candida krusei ATCC 6258) and three filamentous fungi (Aspergillus fumigatus LSI-II, Aspergillus niger ATCC 16404, Aspergillus flavus MTCC 2799). The ATCC cultures used for this study were purchased from American Type Culture Collection, Manassas, VA 20108 USA. RPMI supplemented with 0.165 M MOPS was used as test media. The MIC (Minimum Inhibitory Concentration) was determined by serial 2-fold dilution of the test compound in the above-mentioned media in 100 µL volume in a 96 well U bottom microtitre plate. Yeast inoculums were prepared by growing isolates on Sabouraud Dextrose Agar plates overnight at 37°C. The isolated colonies were picked up and suspensions

ISMAIL et al.: HYDROXYMOYL CHLORIDES AS ANTIFUNGAL AGENTS

were prepared in sterile normal saline with 0.05% (vol/vol) Tween 80 (NST). The density of these suspensions was adjusted to 1 McFarland (1-5 × 106 CFU/mL), further diluted to 1:50 in NST and 1:20 in RPMI 1640 media with 0.165 M MOPS to get 2 times the final inoculum (1-5 × 103 CFU/mL). For filamentous fungi, the inoculums were prepared from the spores of the cultures, which were sporulated on Potato Dextrose Agar (PDA) after an incubation of 7 days at 28ºC. The density of the spore suspension was adjusted to an optical density of 0.09 to 0.11. These suspensions were diluted 1:50 in RPMI 1640 media with 0.165 M MOPS to get the final inoculum (0.4 × 104 to 5 × 104 CFU/mL). 100 µL of this 2 × inoculum of yeast and fungi was added to each well of the microtitre plate. The plates were incubated at 37°C for 48 hr. The plates were read visually and the minimum concentration of the compound showing no turbidity was recorded as MIC. The MFC was determined by spotting 10 µL volume on Sabouraud Dextrose Agar plate from the wells showing no visible growth. The plates were incubated at 37ºC for 48 hr (ref. 18,19). Minimum concentration of compound showing absence of growth was recorded as MFC. Amphotericin-B was taken as a standard. Experimental Section All chemicals (reagent grade) used were commercially available. Melting points were measured on a Buchi D-545 melting point apparatus and are uncorrected. IR spectra were recorded on a Bruker Vector 22 instrument using KBr pellets and in chloroform. 1H NMR were recorded on a Bruker DPX 200 instrument in CDCl3 using TMS as internal standard for protons. 1H NMR chemical shifts and coupling constants J are given in ppm and Hz respectively. Mass spectra were recorded on EIMS (Shimadzu) instrument. Mass-spectrometric (MS) data is reported in m/z. Elemental analysis was carried out using Elemental Vario EL III elemental analyser. Elemental analysis data is reported in % standard. Silica gel 230-400 mesh was supplied by Loba Chemie. Homogeneity of the compounds was checked by TLC on 2 × 5 cm pre-coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck). The chromatograms were visualized under UV 254-366 nm. Typical procedure for the synthesis of 2,3dimethoxybenzaldoxime, 2q: In a typical procedure hydroxylamine hydrochloride (0.50 g, 7.22 mmole) was dissolved in water and neutralized with NaOH.

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To the neutralized solution of hydroxylamine hydrochloride, 2,3-dimethoxybenzaldehyde (1.00 g, 6.02 mmole) was added and the reaction mixture was stirred for 1 hr at RT. Excess of water was added to the reaction mixture and the organic compound was extracted with ethylacetate (2 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to afford pure oxime (syn and anti) in 99% yield. Typical procedure for the synthesis of 2,3dimethoxy phenyl hydroxymoyl chloride, 3q: 2,3Dimethoxybenzaldoxime (1.00 g, 5.52 mmole) was dissolved in DMF (20 mL). N-chlorosuccinimide (0.95 g, 7.18 mmole) was added to the above solution and the reaction-mixture was stirred for 8-10 hr. Excess of water was added and extracted with diethylether (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under vacuum to afford pure chlorooxime (syn and anti). The chlorooxime so formed was subjected to column chromatography (silica gel 230400 mesh as stationary phase, hexane/ethylacetate as mobile phase) and both geometrical isomers were isolated in pure form. Phenyl hydroxymoyl chloride, 3a: (white solid); m.p. 50°C; IR (KBr): 3210.53, 3061.35, 2927.53, 1706.75, 1654.93, 1493.41, 1446.39, 1387.57, 1236.53, 1181.87, 1101.72, 935.50, 763.39 and 691.90 cm-1; 1H NMR (CDCl3): δ 7.42 (3H, m, Ar-H), 7.85 (2H, m, Ar-H); ESI-MS: m/z 155.58 (M+). Anal. Calcd. for C7H6ClNO: C, 54.04; H, 3.89; N, 9.00. Found: C, 53.98; H, 4.00; N, 9.11%. Thiophene-2-hydroxymoyl chloride, 3b: (brownish solid); m.p. 102°C; IR (KBr): 3321.07, 3106.69, 2923.94, 1649.09, 1597.62, 1422.54, 1237.42, 993.23, 877.32, 857.10, 836.72, 800.16 and 710.33 cm-1; 1H NMR (CDCl3): δ 2.13 (1H, s, N-OH), 6.88 (2H, m, Ar-H), 7.05 (1H, m, Ar-H); ESI-MS: m/z 184.61 (M+Na). Anal. Calcd. for C5H4ClNOS: C, 37.16; H, 2.49; N, 8.67. Found: C, 36.97; H, 2.44; N, 8.99%. 4-Chloro phenyl hydroxymoyl chloride, 3i: (white solid); m.p. 76-77°C; IR (KBr): 3292.05, 2924.02, 2852.54, 1650.53, 1595.24, 1488.68, 1401.63, 1245.38, 1093.31, 1014.93, 936.59, 828.95 and 665.98 cm-1; 1H NMR (CDCl3): δ 1.70 (1H, s, NOH), 7.38 (2H, d, J = 8.74 Hz, Ar-H), 7.80 (2H, d, J = 8.77 Hz, Ar-H); ESI-MS: m/z 191.03 (M+1). Anal. Calcd. for C7H5Cl2NO: C, 44.24; H, 2.65; N, 7.37. Found: C, 44.39; H, 2.85; N, 7.12%

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INDIAN J. CHEM., SEC B, MAY 2008

4-Methyl phenyl hydroxymoyl chloride, 3m: (yellow solid); m.p. 71.5-72.5°C; IR (KBr): 3384.87, 2924.57, 2360.95, 2341.82, 1655.30, 1558.76, 1387.98, 1097.87, 1019.00, 896.45, 832.93 and 663.91 cm-1; 1H NMR (CDCl3): δ 2.24 (1H, s, N-OH), 2.40 (3H, s, 4-CH3), 7.24 (2H, d, J = 8.67 Hz, Ar-H), 7.42 (2H, d, J = 8.68 Hz, Ar-H); ESI-MS: m/z 169.62 (M+). Anal. Calcd. for C8H8ClNO: C, 56.65; H, 4.75; N, 8.26. Found: C, 56.53; H, 4.99; N, 8.32%. 2,3-Dimethoxy phenyl hydroxymoyl chloride, 3q: (white solid); m.p. 111.8-13°C; IR (KBr): 3252.47, 3020.13, 2975.25, 19998.04, 1578.64, 1477.46, 1424.85, 1320.11, 1221.47, 1173.56, 1004.04, 984.11, 972.00, 768.58 and 738.88 cm-1; 1H NMR (CDCl3): δ 3.86 (3H,s, -OCH3), 3.88 (3H, s, OCH3), 6.93 (2H, d, J = 8.06 Hz, Ar-H), 7.35 (1H, d, J = 7.78, Ar-H); ESI-MS: m/z 215.65 (M+). Anal. Calcd. for C9H10ClNO3: C, 50.13; H, 4.67; N, 6.50. Found: C, 50.25; H, 4.12; N, 6.66%. 2,3-Dimethoxy phenyl hydroxymoyl chloride, 3q (syn): (white solid); m.p.108.8°C; IR (KBr): 3405.15, 3081.34, 2923.19, 2358.08, 2309.30, 1573.95, 1471.44, 1431.18, 1419.14, 1333.27, 1297.75, 1265.69, 1055.33, 1018.42, 1005.68, 931.46, 858.41, 786.97 and 669.36 cm-1; 1H NMR (CDCl3): δ 3.93 (3H, s, -OCH3), 4.02 (3H, s, -OCH3), 6.7 (2H, d, J = 8.06 Hz, Ar-H), 7.1(1H, d, J = 8.01 Hz, Ar-H); ESIMS: m/z 215.65 (M+). Anal. Calcd. for C9H10ClNO3: C, 50.13; H, 4.67; N, 6.50. Found: C, 50.00; H, 4.78; N, 6.39%. 2,3-Dimethoxy phenyl hydroxymoyl chloride, 3q (anti): (white solid); m.p 90.2°C; IR (KBr): 3355.48, 3081.94, 2924.55, 2359.81, 2310.31, 1574.78, 1477.03, 1420.21, 1339.35, 1274.44, 1232.36, 1095.31, 1045.41, 1006.95, 901.59, 836.46, 808.68, 675.46 and 617.49 cm-1; 1H NMR (CDCl3): δ 3.87 (3H, s, -OCH3), 3.98 (3H, s, -OCH3), 6.93 (2H, d, J = 8.93 Hz, Ar-H), 7.12 (1H, d, J = 8.94 Hz, Ar-H); ESIMS: m/z 216.65 (M+1). Anal. Calcd. for C9H10ClNO3: C, 50.13; H, 4.67; N, 6.50. Found: C, 50.28; H, 4.97; N, 6.44%. 2-Naphthyl hyroxymoyl chloride, 3t: gummy liquid; IR (CHCl3): 3238.21, 3058.80, 2925.55, 1701.82, 1630.42, 1602.23, 1503.74, 1404.30, 1271.58, 1185.20, 1125.23, 1095.29, 861.78, 820.19, 750.37, 657.34, 616.48, 581.31 and 474.61 cm-1; 1 H NMR (CDCl3): δ 1.92 (1H, s, N-OH), 7.54 (2H, m, Ar-H), 7.80-7.95 (3H, m, Ar-H), 8.00 (1H, d, J = 8.69 Hz, Ar-H), 8.34 (1H, s, Ar-H); ESI-MS: m/z 229 (M+Na). Anal. Calcd. for C11H8ClNO: C,64.25; H, 3.92; N, 6.81. Found: C, 64.18; H, 3.99; N, 6.65%.

9-Anthracyl hydroxymoyl chloride, 3v: (yellow solid); m.p. 114-15°C; IR (KBr): 3374.73, 3054.03, 2924.61, 2286.76, 1672.39, 1623.60, 1442.59, 1418.93, 1376.02, 1293.92, 1266.64, 1248.18, 1016.85, 955.19, 892.23, 842.21, 779.28, 733.50, 611.38, 592.52 and 539.22 cm-1. 1H NMR (CDCl3): δ 7.51-7.70 (4H, m, Ar-H), 8.04 (2H, d, J = 8.36 Hz, Ar-H), 8.27 (2H, d, J = 8.71 Hz, Ar-H), 8.54 (1H, s, Ar-H); ESI-MS: m/z 278 (M+Na). Anal. Calcd. for C15H10ClNO: C, 70.46; H, 3.94; N, 5.48. Found: C, 70.54; H, 3.88; N, 5.55%. Acknowledgements The authors thank the Director, IIIM Jammu for his sustained interest and constant encouragement. TI, SS and PPS thank CSIR/UGC, New Delhi for the award of fellowship. References 1 Kontoyannis D, Mantadakis E & Samonis G, J Hosp Infect, 53, 2003, 243. 2 Garber G, Drugs, 61(Suppl. 1), 2001, 1. 3 Fidler D F, Emerg Infect Dis, 4, 1998, 169. 4 Oren I, Temiz O, Yalcin I, Sener E & Altanlar N, Eur J Pharm Sci, 7, 1999, 153. 5 Hong C Y, Farmaco, 56, 2001, 41. 6 Macchiarulo A, Constantino G, Fringuelli D, Vecchiarelli A, Schiaffella F & Fringuelli R, Bioorg Med Chem, 10, 2002, 3415. 7 Patterson T F, Lancet, 366, 2005, 1013. 8 Yanaisawa H, Takamura M, Yamada E, Fujita S, Fujiwara T, Yachi M, Isobe A & Hagisawa Y, Bioorg Med Chem Letters, 10, 2000, 373. 9 Jinda D P, Chattopadhaya R, Guleria S & Gupta R, Eur J Med Chem, 38, 2003, 1025. 10 Pillai A D, Rathod P D, Franklin P X, Padh H, Vasu K K & Sudarsanam V, Biochem Biophys Res Commun, 317, 2004, 1067. 11 Mantylla A, Rautio J, Nevalainen T, Vepsalainen J, Juvonen R, Kendrick H, Garnier T, Croftd S L & Jarvinen T, Bioorg Med Chem, 12, 2004, 3497. 12 Huang S, Li R, Connolly P, Xu G, Gaul M D, Emanuel S L, LaMontagnea K R & Greenberger L M, Bioorg Med Chem Letters,16, 2006, 6063. 13 Minutolo F, Antonello M, Bertini S, Rapposelli S, Rossello A, Sheng S, Carlson E K, Katzenellenbogenc J A & Macchiaa M, Bioorg Med Chem, 11, 2003, 1247. 14 (a) Serrano-wu M H, St Laurent D R, Mazzucco C E, Stickle T M, Barrett J F, Vyas D M & Balasubramanian B N, Bioorg Med Chem Letters, 12, 2002, 943; (b) Emami S, Falahati M, Banifatemi A, Moshiri A & Shafiee A, Arch Pharm Pharm Med Chem, 7, 2002, 318. 15 Liu K C, Shelton B R & Howe R K, J Org Chem, 45, 1980, 3916. 16 (a) Johnson W H, Roberts N A & Borkakoti N, J Enzyme Inhib, 2, 1987, 1; (b) Whittaker M & Brown P, Curr Opin

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